Löptien Jana, Vesting Sidney, Dobler Susanne, Mohammadi Shabnam
Molecular Evolutionary Biology Institute of Cell and Systems Biology of Animals, Universität Hamburg, Hamburg, Germany.
Max Planck Institute for Chemical Ecology, Jena, Germany.
Open Biol. 2024 Dec;14(12):240082. doi: 10.1098/rsob.240082. Epub 2024 Dec 4.
Protein quantification is an important tool for a wide range of biological applications. The most common methods include the Lowry, bicinchoninic acid (BCA) and Coomassie Bradford assays. Despite their wide applicability, the mechanisms of action imply that these methods may not be ideal for large transmembrane proteins due to the proteins' integration in the plasma membrane. Here, we investigate this problem by assessing the efficacy and applicability of these three common protein quantification methods on a candidate transmembrane protein: Na, K-ATPase (NKA). We compared these methods with an ELISA, which we newly developed and describe here for the quantification of NKA. The use of a relative standard curve allows this ELISA to be easily adapted to other proteins and across the animal kingdom. Our results revealed that the three conventional methods significantly overestimate the concentration of NKA compared with the ELISA. This is due to the samples containing a heterogeneous mix of proteins, including a significant amount of non-target proteins. Further, by applying the protein concentrations determined by the different methods to assays, we found that variation in the resulting data was consistently low when the assay reactions were prepared based on concentrations determined from the ELISA.
蛋白质定量是广泛生物应用中的一项重要工具。最常用的方法包括洛瑞法、二喹啉甲酸（BCA）法和考马斯亮蓝法。尽管它们具有广泛的适用性，但作用机制表明，由于这些蛋白质整合于质膜中，这些方法对于大型跨膜蛋白可能并非理想选择。在此，我们通过评估这三种常用蛋白质定量方法对一种候选跨膜蛋白——钠钾ATP酶（NKA）的有效性和适用性来研究这个问题。我们将这些方法与一种酶联免疫吸附测定（ELISA）进行了比较，我们在此新开发并描述了这种用于定量NKA的ELISA方法。使用相对标准曲线使得这种ELISA能够轻松适用于其他蛋白质以及整个动物界。我们的结果显示，与ELISA相比，这三种传统方法显著高估了NKA的浓度。这是因为样品中含有多种蛋白质的混合物，包括大量非靶标蛋白质。此外，通过将不同方法测定的蛋白质浓度应用于分析，我们发现，当基于ELISA测定的浓度制备分析反应时，所得数据的变化始终很低。
